The Regulation of Processing within the Constitutive Secretory Pathway
The Proprotein Convertases (PCs) are responsible for the tissue-specific limited proteolysis of multiple polypeptide precursors, generating a large diversity of bioactive molecules (Seidah and Chretien, 1999; Seidah and Prat, 2002). Many cellular processing events involve an ordered cascade of cleavage events accomplished by one or more convertase(s) belonging to the PCs/SKI-1/PCSK9 mammalian subtilase family (Seidah and Chretien, 1999; Seidah and Prat, 2002; Seidah et al., 2003). This mammalian PC-family comprises nine members: PC1/3, PC2, furin, PC4, PACE4, PC5/6, PC7, SKI-1/S1P and NARC-1/PCSK9 (Seidah and Chretien, 1999; Seidah and Prat, 2002; Seidah et al., 2003). The first seven proteinases are basic amino acid specific PCs cleaving precursor proteins at single or paired basic residues within the motif (K/R)—(X)n-(K/R)↓, in which n=0, 2, 4 or 6 (Seidah and Chretien, 1999). These proteinases are phylogenetically more closely related to each other and to yeast kexin than to SKI-1/S1P or NARC-1/PCSK9, which belong to the pyrolysin (Seidah et al., 1999) and proteinase K (Seidah et al., 2003) subfamilies, respectively. The latter enzymes recognize the motifs R—X-(hydrophobic/aliphatic)-Z↓ (Seidah et al., 2006) and VFAQ↓ (SEQ ID NO: 116) (Benjannet et al., 2004), respectively. These enzymes have been implicated in a wide variety of functions regulating cellular homeostasis and a number of pathologies including cancer, inflammation, neurodegenerative diseases, atherosclerosis and viral infections. It was recently realized that some of these convertases play critical roles in the regulation of lipids and/or sterols (Seidah et al., 2006) either through the inactivation of lipoprotein lipases e.g., by PC5/6, PACE4 and furin (Jin et al., 2005), activation of specific membrane-bound transcription factors (SREBP-1 and -2) by SKI-1/S1P (Cheng et al., 1999), or by enhancing the degradation of the low density lipoprotein receptor (LDLR) by PCSK9 (Maxwell and Breslow, 2004; Benjannet et al., 2004; Park et al., 2004; Maxwell et al., 2005).
A number of factors regulate the processing of PCs themselves. First, convertases require removal of their inhibitory prosegment for activation (FIG. 1). Analysis of the biosynthesis of furin, PACE4, PC5, PC7, SKI-1, and PCSK9 revealed that they are synthesized as zymogens that undergo autocatalytic cleavage of their N-terminal inhibitory pro-segment, which seems to act both as a chaperone and an intramolecular inhibitor (Zhong et al., 1999; Nour et al., 2003; Seidah et al., 2003). Except for PC2, primary prosegment cleavage is necessary for most convertases to exit from the endoplasmic reticulum (ER). Overexpression of furin, PC5, and PC7 prosegments as independent domains confirmed their inhibitory potency and the presence of critical elements at their C-terminus. The design of two potent and specific-inhibitors of SKI-1 based on variants of either its prosegment or α1-PDX were reported (Pullikotil et al., 2004).
The second control element is the trafficking of these enzymes to different intracellular organelles. Dependant on the cognate substrate, constitutively secreted PCs cleave in the Golgi, trans Golgi network (TGN), endosomes or at the cell surface (FIG. 2). The modified serpin α1-PDX (Benjannet et al., 1997; Anderson et al., 1993) and the PC-prosegments (Zhong et al., 1999) inhibit the PCs within the constitutive secretory pathway.
Regulation and Processing of PCSK9
The regulation of PCSK9 activity could be achieved by various mechanisms, which among others could act at the level of: (i) its transcription where its mRNA levels are upregulated by SREBP-2, and downregulated by cholesterol (Maxwell et al., 2003; Dubuc et al., 2004) via a reduced level of activated nuclear SREBP-2 (Horton et al., 2003; Dubuc et al., 2004); (ii) its translation which may be controlled by specific factors; (iii) its post-translational modifications including its zymogen cleavage and/or activation, glycosylation, sulfation (Seidah et al., 2003; Benjannet et al., 2004), or possibly by other processing events resulting in its degradation (Seidah et al., 2003); (iv) its cellular localization and/or sorting of mature PCSK9; (v) its level of secretion; and possibly, (vi) its subsequent cellular re-uptake.
PCSK9/NARC-1 plays a role in cholesterol homeostasis. Indeed, point mutations in the PCSK9 gene within its coding exons (Attie, 2004) are associated with either familial hypercholesterolemia (Abifadel et al., 2003; Leren, 2004; Timms et al., 2004; Allard et al., 2005; Naoumova et al., 2005) or hypocholesterolemia (Cohen et al., 2005; Kotowski et al., 2006; Berge et al., 2006) phenotypes. This led to the classification of the PCSK9 gene as the third chromosomal locus associated with autosomal dominant familial hypercholesterolemia, with the LDLR and Apolipoprotein B (Apo B) comprising the other two loci (Abifadel et al., 2003). It is thus plausible that some of the single point mutations of PCSK9 associated with autosomal dominant familial dyslipidemias could enhance or abrogate one or more of the PCSK9 regulatory events (Attie and Seidah, 2005).
It should be noted that PCSK9 is mostly expressed in adult liver hepatocytes and in small intestinal enterocytes (Seidah et al., 2003). Its exact role in these tissues is still unknown, except that this convertase possibly enhances the rate of degradation of the endogenous hepatic and possibly intestinal LDLR. Although no PCSK9 inhibitor is yet known, potent PCSK9 siRNAs were identified that upregulate the LDLR (Benjannet et al., 2004). The lowering of the level of LDLR at the cell surface is thus a good indicator of the PCSK9 activity.
Natural Mutants of PCSK9 and Implication in Hypocholesterolemia
It was suggested that some PCSK9 single point mutations result in a gain or enhanced function of PCSK9 on the degradation of LDLR in acidic compartments, possibly endosomes (Benjannet et al., 2004; Maxwell et al., 2005), while others would cause a loss of function (Cohen et al., 2005), and would be associated with the development of hyper- or hypo-cholesterolemia, respectively (Attie and Seidah, 2005; Kotowski et al., 2006). It was thus hypothesized that high levels of active PCSK9 are associated with a faster rate of degradation of the cell surface LDLR, resulting in increased amounts of circulating LDL-cholesterol, as the uptake of the latter in liver hepatocytes by the LDLR will be diminished accordingly, and vice versa. This would suggest that the level of cell surface LDLR is indirectly proportional to the level of hepatic and likely intestinal active PCSK9. This hypothesis is reinforced by the in vivo observations that in mice lacking a functional PCSK9 gene (PCSK9-knockout mice), the level of hepatocyte cell surface LDLR is greatly enhanced resulting in an ˜50% drop in the level of circulating LDL-cholesterol (Rashid et al., 2005), whereas mice overexpressing PCSK9 result in higher levels of circulating LDL-cholesterol (Benjannet et al., 2004; Park et al., 2004; Maxwell and Breslow, 2004; Lalanne et al., 2005).
Examples of hypercholesterolemic-associated mutations include the Ser127-to-Arg (S127R) amino acid change. The S127R mutation is located between the primary and putative secondary zymogen processing sites of the PCSK9 propeptide; mutations in the catalytic subunit include Phe216-to-Leu (F216L), which is located close to the active site at His226 (Abifadel et al., 2003) and Arg218-to-Ser (R218S) (Allard et al., 2005). These and other new natural mutations reported in Table 1 below were biochemically analyzed and some of them were suggested to result in a gain of function, likely including a gain of proteolytic activity or a better co-localization with LDLR (Benjannet et al., 2004). However, the molecular mechanisms that underlie the dominance of the dyslipidemia trait caused by PCSK9 missense mutations is still unclear.
Regulation of Other Cell Surface Receptors by PCSK9
Only the LDLR was reported to be affected by PCSK9. The LDLR is part of the 7-member LDL receptor superfamily that included amongst others, very low density lipoprotein receptor (VLDLR) (Official Symbol: VLDLR and Entry gene ID: 7436, NCBI), apolipoprotein e receptor 2 (ApoER2) (Official Symbol: LRP8 and Entry gene ID: 7804, NCBI) and LRP (FIGS. 11 and 12). LRP, a member of the 7-member LDL receptor superfamily that includes amongst others, LDLR, very low density lipoprotein receptor (VLDLR) (Official Symbol: VLDLR and Entry gene ID: 7436, NCBI), apolipoprotein e receptor 2 (ApoER2) (Official Symbol: LRP8 and Entry gene ID: 7804, NCBI) (FIGS. 11 and 12), was found not to be affected by PCSK9 (Benjannet et al., 2004). There is a need to develop a cell system that would allow the identification of novel PCSK9 targets.
Implication of PCSK9 in Human Pathologies
PCSK9 has clearly been involved in the regulation of LDL-cholesterol. Dyslipidemia is in fact the first dominant human pathology directly associated with mutations in a PC, namely in PCSK9. Since PCSK9 is also expressed in brain and gut (Seidah et al., 2003), it is plausible that mutations in PCSK9 may have other consequences aside from LDL-cholesterol regulation. Such pleiotropic effects were reported for other convertases. An example includes the β-secretase BACE1, which has been clearly implicated in Alzheimer's disease, but whose functions seems also to include memory and emotion regulation independent of its effect on the processing of β-amyloid precursor (Laird et al., 2005).
A definition of novel functions of PCSK9 would alert to potential mechanism-based side effects that may occur with PCSK9 inhibitors designed to decrease LDL-cholesterol levels. Thus, a sensitive assay for PCSK9 function is urgently needed, which may uncover new unsuspected functions of this enzyme.
In Vitro PCSK9 Assays
Most of the in vitro assays designed for identifying proteinase inhibitors consist in the addition of the compound to a reaction mixture containing a purified enzyme and its substrate, and measuring the absence or reduction of the cleavage products observed when the mixture is incubated under similar conditions but without the inhibitory compound. However, since none of the existing methods allowed for the detection of an active enzymatic form of PCSK9, no such in vitro assays are yet available using PCSK9 for identifying PCSK9 inhibitors. Furthermore, some inhibitors active in vitro may not find utility in vivo because of their inability to enter the cell and reach the cellular compartments where PCSK9 is localized. There is thus a need for the development of cell-based assays specific for PCSK9 activities.
PCSK9 Cell-Based Assays
Prior art cell-based assays for identifying convertase-inhibitory compounds produce false positives. For instance, Oh et al. 2004 described a cell-based assay for β-secretase activity using a target chimeric protein substrate containing three domains: an amino-terminal TM domain, a beta-site and an alkaline phosphatase (AP). In this assay, the activity of BACE on the chimera results in the release of AP in the culture medium. An inhibition of the BACE activity results in the absence of AP release in the culture medium. An absence of AP in the culture medium could result not only from the inhibition of the target substrate synthesis itself, but also from a variety of irrelevant cellular mechanisms including amongst others, the absence of target chimeric protein substrate expression itself, modification of chaperones, cellular trafficking, protein folding or even a pH change within the cells, etc. It is thus difficult to determine through their use whether the absence of detection of a specific signal resulted from enzyme inactivation or from another irrelevant reason.
Although a positive cell-based assay which targets cathepsin L in the lysosome and used for the identification of protease inhibitors was described (Belkhiri et al., 2002), this assay is not appropriate for the identification of PCSK9 inhibitors. Other positive cell-based assays measuring the increase of a signal molecule at the cell surface do not offer the appropriate sensitivity for screening due to the high background of the signal molecule already present at the cell surface. For instance, the measurement of re-appearance of LDLR at the surface of cells overexpressing wild type PCSK9 does not provide a sensitive positive screening for PCSK9 inhibitory compounds due to the LDLR background that still remains at the cell surface (Benjannet et al., 2004).
There is thus a need for an improved positive cell-based assay adapted to PCSK9.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.